Methods and compositions for preparation of double stranded nucleic acid molecules, e.g. dsDNA, by novel connectors that include linkers that attach the molecules to a matrix, are useful for the investigation of protein-nucleic acid interactions and nucleic acid structure.
Single-stranded DNA (ssDNA) microarrays have been extensively reported and used to screen genetic polymorphisms, mutations and gene expression to answer various questions. However, analysis of genetic transcription, replication, and restriction enzyme engineering requires dsDNA microarrays.
During the past several years many developments were reported in DNA microarray (biochip)-based automated technology that allowed parallel analysis of multiple DNA samples. Most of these biochips are based on application of single stranded DNA (ssDNA) microarrays, which are used in hybridization experiments with different kinds of DNA samples. Single-stranded DNA biochips are reported for gene expression profiling, gene polymorphism analysis, and mutation screening.
However, many important investigations in molecular biology cannot be served by this format of DNA microarrays. For example, systematic investigation of DNA-protein interactions which includes investigation of recombination, transcription, replication, and also discovery and engineering of new restriction enzymes, requires double stranded DNA microarrays.
There are many different approaches to the creation of double stranded DNA (dsDNA) microarrays. Different supports, linkers, base pair lengths, and types of DNA are reported for various applications.
A brief review of the major work and achievements in dsDNA biochip manufacturing is as follows:
There were two main approaches to construct dsDNA arrays. In the first, a single oligonucleotide strand was attached to a matrix, and then duplexes were formed by hybridization of the attached strand with complementary chains. The second method is based on manufacturing dsDNA arrays by using hairpin stem-loop DNA molecules attached to the matrix across the loop-part and hybridized to form only a partial duplex structure. The full duplex can be obtained by using enzyme polymerase if needed.
A method used for fabrication of dsDNA microarrays on a glass surface is illustrated in FIG. 1 (Bulyk, 1999). In the first step of creation of dsDNA arrays, single stranded DNA probes are synthesized on a glass surface by light-directed methods of oligonucleotide synthesis. Unfortunately, the efficiency of this method of synthesis is not high—92–94% per one step of oligonucleotide synthesis, thus for a 40-mer oligonucleotide, only from 5 to 20% of the synthesized oligonucleotides on the glass surface will be of the desired length and sequence (Carlson, 1999). Short synthetic primers are then annealed to the single stranded oliogonucleotide probes on the glass surface. Finally, the full duplex is obtained by Klenow polymerase reaction. The labeling of dsDNA probes thus created is carried out by fluorescein labeled dNTPs during a polymerization reaction, or by use of terminal transferase addition of fluorescein-labeled ddNTPs. However, this method is very time consuming and it is impossible to guarantee the high precision of answers using these microarrays.
Another approach to manufacturing dsDNA arrays is shown in FIG. 2. In this method (Braun, 1998) the first step uses the same light-directed oligonucleotide synthesis as in FIG. 1 on a glass or gold surface e.g. a microscope “slide.” Then, double stranded DNA fragments with protruding ends (complementary to probes attached to the chip) are hybridized to the immobilized oligonucleotides. The treatment of the resulting complex with DNA ligase creates a surface coupled dsDNA microarray. However, all disadvantages of the previously described method are present here too.
Another way for obtaining dsDNA arrays was demonstrated by O'Brien and coworkers (see FIG. 3). The authors made dsDNA microarrays for protein—dsDNA screening and investigation of antigen-antibody binding using Atomic Force Microscopy. First, the set of complementary oligonucleotide pairs was synthesized by means of a standard solid phase approach using phosphoramidite chemistry (Sinha, 1983). Then, self-assembled DNA duplexes with recognition sites for the EcoRI restriction endonuclease and fluorescein moieties above the recognition sequences were obtained by annealing complementary chains in solutions before the creation of microarrays. Each of the complementary oligonucleotides contained a disulfide moiety, which provided further bonding of the duplexes to the surface of slide. However, the authors emphasized that there were serious problems with the standardization of capacity parameters of microarrays in that method. Also the high level of nonspecific adsorption of DNA probes can dramatically affect further experiments with this chip and interpretation of obtained results.
Other methods created DNA microarrays with hairpin structured probes (see FIG. 4A, Zhao et al., 2001). One method was based on the covalent attachment of the hairpin stem-loop structure to a matrix. In this work oligonucleotides with five phosphorothioate residues in the loop were synthesized. The presence of these multiple phosphorothioate functions in the hairpin structure was used to anchor the oligonucleotide to a glass slide surface. A problem was that covalent attachment to the slide may occur statistically in every position of the loop, so geometric parameters of the hairpin can vary and as a result, can influence the formation of correct duplexes, especially for short complements (e.g. 5 base pairs). Then, complete labeled duplexes were obtained by extension of hairpin structures on a glass slide by use of T7 sequenase in the presence of cyanine dye labeled ddGTP (see FIG. 4B)
The paper of Riccelli and coworkers (see FIG. 5A) reported the use of hairpin probes made from a partial duplex (16 base pairs and a 32-base long single strand end) and a loop with biotinylated uracil as a linker in the middle. By this linker the structure was coupled to avidin coated microtiter wells. It was reported that such hairpin DNA probes attached to the chip displayed higher rates of hybridization and larger equilibrium amounts of captured targets in comparison with linear probes. Also, hairpin DNA—target complexes were thermodynamically more stable.
DNA microarrays containing stem-loop DNA probes with short single-stranded overhangs were immobilized on 3-dimensional Packard HydroGel chips by Broude et al. (2001). Microarrays were fabricated by immobilizing pre-synthesized, self-complementary single-stranded oligonucleotides which adopt partially duplex dsDNA was then biotinylated at single stranded regions. The biotinylated dsDNA was then used as a ligand at a gold electrode covered by avidin. The obtained biosensors were said to be useful to determine small molecular weight organics, that is, a dsDNA based sensor, and for monitoring DNA-analyte interactions.
Although much effort has been expended in this research area, improvements are needed so that dsDNA microchips can be manufactured efficiently and used effectively.